PROJECT SUMMARY/ABSTRACT: N6-methyladenosine (m6A) is a reversible modification in mRNAs that has been shown to regulate many aspects of mRNA metabolism and function, including splicing, translation, and degradation. The biological effects of m6A methylation are mediate by writer enzymes that methylate mRNA, eraser enzymes that remove the methylation mark, and reader enzymes that selectively bind m6A residues. m6A methylation is involved in a number of important biological processes, including regulating mRNA turnover during stem cell differentiation. Despite its importance as a gene regulatory mechanism, how mRNA methylation influences the development and pathology of cancer is not well understood. In particular, comprehensive characterization of m6A levels and the roles of the writer, eraser, and reader proteins in cancer patients is lacking. Cancer sequencing efforts have found that METTL14, a subunit of the m6A writer complex, is frequently mutated in patients with endometrial cancer, providing an opportunity to investigate the role of m6A methylation in cancer. Preliminary studies of endometrial tumors suggest that reduced m6A methylation in tumors may act as a novel, key driver of tumor growth and malignancy. Therefore, I propose to extend these preliminary data by performing experiments to investigate the role of reduced m6A methylation as a driver of endometrial cancer, determine the mechanisms for how reduced m6A methylation alters the proliferation of endometrial cancer, and investigate the factors leading to altered m6A methylation in endometrial tumors. In Aim 1, I will characterize the effects of writer knockdown and eraser overexpression in endometrial cancer cell lines to determine how reduced m6A methylation affects cell proliferation in vitro and in vivo. In addition, I will characterize these cell lines as well as tumor samples exhibiting altered m6A methylation to identify particular transcripts and mechanisms mediating the changes to cell proliferation. In Aim 2, I will investigate which signaling pathways commonly altered in endometrial cancer regulate m6A methylation in endometrial cancer. I will supplement these experiments with sequencing studies of patient samples to identify additional factors that regulate m6A methylation in endometrial cancer. Together these experiments will advance our understanding of the mechanisms underlying endometrial cancer, provide insights into the signaling pathways that regulate mRNA methylation, and may establish these key enzymes in the mRNA methylation pathway as targets for novel cancer diagnostics and therapeutics.